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Field observations show that velvet bentgrass (Agrostis canina L.) (VB) an alternative species to creeping bentgrass (Agrostis stoloniera L.) (CB) exhibits chlorosis in the leaves at high nitrogen rates. Growth chamber experiments were conducted that compared VB and CB using a hydroponic system. The experiments tested 5%NH4+-N, 45%NH4+-N, 95%NH4+-N and urea N-sources. Chlorosis only occurred in VB at the 95% NH4+-N and 100% urea-N source. Nitrate, nitrite, and ammonium levels in leaves showed ammonium accumulation was positively correlated to chlorosis in VB. Enzyme analysis showed initial glutamine synthetase (GS) activity in roots compared to leaves is higher in CB than in VB, explaining low ammonium levels in leaves of CB. The GS activity in the roots of VB increased after 24 hours of treatment. The results showed that the chlorosis in the leaves of VB is the correlated to ammonium accumulation associated with nitrogen form supplied and GS activity.